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2.6.4 Summary statement of the pharmacokinetic study 





TABLE OF CONTENTS 
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LIST OF TABLES 


Table 1 Pharmacokinetics of luciferase RNA-encapsulated LNPs, ALC-0315 and ALC- 
0159, when administered intravenously to Wistar Han rats at a dose of 1 mg 
FUP crass pccpassdoeas tesa tardtaontpianratosusdsevqnisesiea’ ciutertoyinmnsatnatirsesnas pases iAaaemmaeatneinenerasieh 4 
LIST OF FIGURES 
Figure 1 Plasma and liver concentrations of ALC-0315 and ALC-0159 after intravenous 
administration of luciferase RNA-encapsulated LNPs at a dose of 1 mg RNA/kg to Wistar 
MTN lite ss Apaetsestbcoseslster!saacida cetececsttestaeeceleenctenarcanacte vente secebiaat stediatesectisectataceatesin pialusectitaveuersisiacacties 5 
Figure 2 In vivo luminescence in BALB/c mice intramuscularly treated with luciferase 
BeAr Ce SUIS CLIN IP fences cient cance ta ceaade selec snadsehadeteacai tea ieshoensaeeascicoddaenaaqesacasenanerctauienudsciaumtnccis 6 
Figure 3 Estimated in vivo metabolic pathways of ALC-0315 in various animal species..8 


Figure 4 Estimated in vivo metabolic pathways of ALC-0159 in various animal species. 9 


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SARS-CoV-2 mRNA Vaccine (BNT162, PF-07302048) 


2.6.4 Summary statement of the pharmacokinetic study 





Terms and abbreviations used in this section 





Terms and abbreviations 


Unabridged expressions or definitions 















































ALC-0159 PEG lipids added to the drug 

ALC-0315 Aminolipids added to this product 

[sH]-CHE Radiolabeled [Cholesteryl-1,2-3H(N)]-Cholesteryl Hexadecyl Ether 

DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine 

GLP Good Laboratory Practice 

LNP Lipid-nanoparticle 

modRNA Nucleoside-modified mRNA 

mRNA Messenger RNA 

m/z m/z 

PEG Polyethylene glycol 

PK Pharmacokinetics 

RNA Ribonucleic acid 

S9 Supernatant fraction obtained from liver homogenate by 
centrifuging at 9000 g 

WHO World Health Organization 








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2.6.4 Summary statement of the pharmacokinetic study 





1. Summary 

BNT162b2 (BioNTech code: BNT162, Pfizer code: PF-07302048) is a modified nucleoside 
mRNA (modRNA) encoding the full-length spike glycoprotein (S protein) of severe acute 
respiratory syndrome coronavirus 2 (SARS-CoV-2). It is being developed as the essence 
of an MRNA vaccine against infections caused by SARS-CoV-2. To formulate BNT162b2, 
it was mixed with two functional lipids, ALC-0315 (aminolipid) and ALC-0159 (PEG-lipid), 
and two structural lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and 
cholesterol. (1,2-distearoyl-sn-glycero-3-phosphocholine) as two structural lipids, and 
cholesterol to form lipid nanoparticles (LNPs) that encapsulate BNT162b2 (henceforth, 
"BNT162b2 encapsulated LNPs"). 


To evaluate the non-clinical pharmacokinetics of BNT162b2-encapsulated LNPs, we 
performed evaluation studies both in vivo and in vitro to see the absorption (PK), 
metabolism, and excretion of ALC-0315 and ALC-0159 in LNPs, as well as biodistribution 
studies using luciferase or radioactively labeled lipid as alternative reporters for 
BNT162b2. 


Based on the fact that the development of vaccines for the prevention of infectious 
diseases does not require evaluation of systemic exposure (WHO, 2005; Guidelines for 
Non-clinical Studies of Vaccines for the Prevention of Infectious Diseases) 1,2, we did not 
conduct a PK study using intramuscular administration of BNT162b2-encapsulated LNP. 
The other two lipids contained in the drug (cholesterol and DSPC) are naturally occurring 
lipids and are considered to be metabolized and excreted in the same manner as 
endogenous lipids. In addition, it is expected that BNT162b2 is degraded by ribonuclease 
in uptake cells and metabolized by nucleic acid, and that BNT162b2-derived S protein 
undergoes proteolysis. Based on the above, it was not considered necessary to evaluate 


the metabolism and excretion of these components again. 


As an alternative reporter to BNT162b2, luciferase RNA-encapsulated LNPs (luciferase 


RNA encapsulated in LNPs with the same lipid composition as BNT162b2-encapsulated 


LNPs; hereafter referred to as "luciferase RNA-encapsulated LNPs") were intravenously 
administered to Wistar Han rats. In the study, plasma, urine, feces, and liver samples 
were collected over time, and the concentrations of ALC-0315 and ALC-0159 were 
measured in each sample. The results showed that ALC-0315 and ALC-0159 were rapidly 
distributed from the blood to the liver. About 1% and 50% of the dose of ALC-0315 and 
ALC-0159, respectively, were excreted in the feces as unchanged drug, and both were 


below the detection limit in the urine. 


In the biodistribution study, luciferase RNA-encapsulated LNP was intramuscularly 
administered to BALB/c mice. In the biodistribution study, luciferase RNA-encapsulated 
LNP was intramuscularly administered to BALB/c mice, and the expression of luciferase 
was observed at the site of administration and also in the liver where the level of 
expression was even lower. Expression of luciferase at the site of administration was 
observed at 6 hours post-dose and disappeared by 9 days post-dose. Expression of 
luciferase in the liver was also observed at 6 hours post-dose and disappeared by 48 
hours  post-dose. The radioactivity of the radioactively labeled luciferase RNA- 
encapsulated LNPs was intramuscularly administered to rats, and the biodistribution of 
the radioactivity was quantitatively evaluated. The highest non-dose site was the liver 
(up to 18% of dose). 


The metabolism of ALC-0315 and ALC-0159 was evaluated in vitro using blood, liver 
microsomes, liver S9 fractions and hepatocytes from CD-1/ICR mice, Wistar Han or 
Sprague Dawley rats, crab-eating macaques or humans. In vivo metabolism was also 
investigated using plasma, urine, feces, and liver samples collected from the intravenous 
PK study in rats. These in vitro and in vivo studies showed that ALC-0315 and ALC-0159 
were slowly metabolized by hydrolysis of ester and amide bonds, respectively, in all 


animal species tested. 


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2.6.4 Summary statement of the pharmacokinetic study 





These non-clinical pharmacokinetic evaluations indicated that LNP that reached the 
circulation was distributed to the liver. In addition, the disappearance of ALC-0315 and 


ALC-0159 was suggested to be related to metabolism and fecal excretion, respectively. 


2. Analysis method 

Report Number: PF-07302048_06 72424 

An LC/MS method with appropriate performance was developed to quantify the 
concentrations of ALC-0315 and ALC-0159, the constituent lipids of LNP, in the non-GLP 
rat intravenous PK study (Section M2.6.4.3). In other words, 20 wL of plasma, liver 
homogenate (homogenates were prepared from sections taken from three different 
locations of the liver and pooled and diluted with blank matrix as appropriate), urine and 
fecal homogenate (diluted with blank matrix as appropriate) samples were each 
deproteinized in acetonitrile containing an internal standard (PEG-2000). The samples 


were centrifuged and the supernatant was used for LC-MS/MS measurements. 


3. Absorption 

Report Number: PF-07302048_06 72424, Summary Table: 2.6.5.3 

To investigate the pharmacokinetics of ALC-0315 and ALC-0159, luciferase RNA- 
encapsulated LNPs were administered intravenously to male Wistar Han rats at a single 
dose of 1 mg RNA/kg, and plasma and liver samples were collected by sparse sampling 
over time (0.1, 0.25, 0.5, 1, 3, 6, and 24 h before, and 2, 4, 8, and 14 days after 
administration). Plasma and liver samples were collected by sparse sampling (3 
animals/time point). Plasma and liver concentrations of ALC-0315 and ALC-0159 were 
measured and PK parameters were calculated (Table 1). ALC-0315 and ALC-0159 in 
blood were promptly distributed to the liver by 24 hours after administration. Plasma 
concentrations of ALC-0315 and ALC-0159 at 24 hours post-dose were less than 1% of 
the maximum plasma concentration (Figure 1). The apparent terminal phase elimination 
half-lives (t%) were similar in plasma and liver, 6-8 days for ALC-0315 and 2-3 days for 
ALC-0159. The results of this study suggest that the liver is one of the major tissues that 
take up ALC-0315 and ALC-0159 from the blood. 


The results of the investigation of urinary and fecal concentrations of ALC-0315 and ALC- 
0159 conducted in this study are described in section M2.6.4.6. 


Table 1 Pharmacokinetics of ALC-0315 and ALC-0159 in LNP included with luciferase labeled RNA injected 
intravenously in Wistar Han Rat at Img RNA/kg 





component Amountofcomponent Sex/N t% (h) AUCint AUCaast Distribution in 
(mg/kg) (ugeh/mL) (ug*h/mL) the Liver (%) 
ALC-0315 15.3 Male/3> 139 1030 1020 60 
ALC-0159 1.96 Male/3> 72.7 99.2 98.6 20 


a. Calculated by [Max amount of distribution in the Liver]/[Total amount injected] 
b. 3 animals in each point. Spars sampling 


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2.6.4 Summary statement of the pharmacokinetic study 





Figure 1 Plasma and liver concentrations of ALC-0315 and ALC-0159 after intravenous 


administration of luciferase RNA-encapsulated LNPs at a dose of 1 mg RNA/kg to Wistar 
Han rats 





=—@— ALC-0315. inplasma : =— @ — ALC-0315| in the Liver 
6 = @ = ALC-0159) in the Liver 
10% —e— ALC-0159 in plasma is ? O~n g_ se cai 
O -_— 
b =O --e---- © 
tu) 
E D> 10 & 
= = \ 
Cc c a 
B=] 2 ‘ 
£ 01 g *. 
c fo ~ 
8 8 i — 
36) fo) ap 
O 1S) 
001 
) 100 200 300 0 100 20 yx 
Time after injection (h) Time after injection (h) 


4. Distribution 

Report No.: REoo72, 185350, Summary Table: 2.6.5.5A, 2.6.5.5B 

Luciferase RNA-encapsulated LNPs were administered to three female BALB/c mice, 
and the biodistribution of BNT162b2 was examined using luciferase luminescence as a 
surrogate marker. In other words, luciferase RNA-encapsulated LNPs_ were 
intramuscularly administered to the left and right hind paws of mice at a dose of 1 wg 
RNA each (2 wg RNA in total). Luciferin, a luminescent substrate, was then administered 
intraperitoneally 5 min before detection of luciferase luminescence, and in vivo 
luminescence was measured under isoflurane anesthesia at 6 and 24 hours after 
administration and at 2, 3, 6, and 9 days using Xenogen IVIS Spectrum. The expression 
of luciferase protein was evaluated over time in the same individual. The expression of 
luciferase at the site of administration was observed from 6 hours post-dose and 
disappeared by 9 days post-dose. The expression of luciferase in the liver was also 


observed from 6 hours post-dose and disappeared by 48 hours post-dose. The 


distribution in the liver was considered to indicate that a portion of the locally 
administered luciferase RNA-encapsulated LNP reached the circulating blood and was 
taken up by the liver. As described in detail in Section M2.6.4.3, when luciferase RNA- 
encapsulated LNPs were administered intravenously to rats, the liver was suggested to 
be the major organ of distribution for ALC-0315 and ALC-0159, which is consistent with 
the findings of this study in which ALC-0315 and ALC-0159 were administered 
intramuscularly to mice. No toxicity findings indicating hepatic injury were observed in 


the rat repeated-dose toxicity study (Section 2.6.6.3). 


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2.6.4 Summary statement of the pharmacokinetic study 





Figure 2 In vivo luminescence in BALB/c mice intramuscularly treated with luciferase 


6h 


24h 


6d 





RNA-encapsulated LNP 


10*° 


10° 


¢ + 


10° 
10’ 
10° 
10° © o—_e——_ ° 


10 


Luciferase RNA-encapsulated LNPs using [3H]-cholesteryl hexadecyl ether ([3H]-CHE)- 
labeled LNPs were administered intramuscularly to male and female Wistar Han rats at 


a dose of 50 wg RNA, and blood, plasma, and tissues were collected from three animals 
per sex at 15 min and at 1, 2, 4, 8, 24, and 48 h after administration. Blood, plasma, and 


tissue samples were collected from three animals each at 15 min and 1, 2, 4, 8, 24, and 


48 h post-dose, and the biodistribution of LNPs was evaluated by measuring the 


radioactivity concentration by liquid scintillation counting. In both males and females, the 


highest radioactivity levels were observed at the site of administration at all 
measurement time points. Plasma radioactivity levels were highest in the first 1 to 4 
hours post-dose. The highest radioactivity levels in these tissues were observed 8 to 48 
hours post-dose. Total recoveries of radioactivity relative to the dose outside of the dose 
site were highest in the liver (up to 18%) and were significantly lower in the spleen 
(<1.0%), adrenal glands (<0.11%), and ovaries (<0.095%) than in the liver. The mean 
radioactivity concentrations and tissue distribution patterns were generally similar in 


males and females. 


The distribution of the antigen encoded by BNT162b2 in vivo is considered to be 
dependent on the distribution of LNP. Since the lipid composition of the luciferase RNA- 
encapsulated LNP used in this study is identical to that of the application formulation of 
BNT162b2, the results of this study are considered to indicate the distribution of 
BNT162b2-encapsulated LNP. 


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2.6.4 Summary statement of the pharmacokinetic study 





5. Metabolism 

Report Number: 01049 008, 01049 009, 01049f{ 010, 0104 020, 01049 021, 01049 
022, PF-07302048 05 flo43725, Summary Table: 2.6.5.10A, 2.6.5.10B, 2.6.5.10C, 
2.6.5.10 D 

The in vitro metabolic stability of ALC-0315 and ALC-0159 was evaluated using liver 
microsomes, liver S9 fractions, and hepatocytes from CD-1/ICR mice, Wistar Han or 
Sprague Dawley rats, crab-eating macaques, and humans. ALC-0315 or ALC-0159 were 
added to liver microsomes or liver S9 fractions (120 min incubation) or hepatocytes (240 
min incubation) of each animal species, and the percentage of unchanged product after 
incubation was determined. The results showed that ALC-0315 and ALC-0159 were 
metabolically stable in both animal species and test systems, and the final percentage 


of unchanged product was over 82%. 


In addition, the metabolic pathways of ALC-0315 and ALC-0159 were evaluated in vitro 
and in vivo. In these studies, in vitro metabolism was evaluated using blood, liver S9 
fractions, and hepatocytes from CD-1 mice, Wistar Han rats, crab-eating macaques, and 
humans. In addition, plasma, urine, feces, and liver samples collected in the rat PK study 
were used to evaluate in vivo metabolism (Section M2.6.4.3). The test results showed 
that the metabolism of both ALC-0315 and ALC-0159 was slow and metabolized by 
hydrolysis of ester and amide bonds, respectively. Metabolism by hydrolysis, shown in 


Figure 3 and Figure 4, was observed in all animal species evaluated. 


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2.6.4 Summary statement of the pharmacokinetic study 





Figure 3 The estimated metabolic pathway in organisms of ALC-0315 
in various spices of animals 


} 1O-~ yy eee eee 


a O 
'S OOCe 
ALC-0815 
in blood (mo, r) Poe iad ad i 
in hepatocytes(mo, r, mk,h) oe 
in liver s9 (mo, r, h) een cw 
in plasma (r) cata Gediccls adiaastaaial in blood (mo, r) 
‘ in liver s9 (mk) 
o in plasma (r) 
in blood (mo, r) cw m/z 528 in liver (r) 
in hepatocytes(mo,r,mk,h) "Oy ~~~ ~ 
in liver s9 (mo, r, h) m/z 255 
in plasma (r) ee anal in blood (mo, r) 
aL in liver s9 (mk) 
ee in plasma (r) 
mz 200 in urine (r) 
in feces (r) 
in liver (r) 
glucuronide 
Ona Qn OH 
u 
M in urine (r) 
Ou 
m/z 466 


H: Human, Mk: Monkey, Mo: Mouse, R: Rat 


ALC-0315 is metabolized by undergoing two successive rounds of ester hydrolysis. These 
two hydrolysis events produce first the monoester metabolite (m/z 528) and then the 
double transesterification metabolite (m/z 290). The double transesterification 
metabolite was further metabolized to a glucuronide conjugate (m/z 466), which was 
detected only in urine in the rat PK study. It was also confirmed that the acidic products 
of the two hydrolyses were both 6-hexyl decanoic acid (m/z 255). 


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2.6.4 Summary statement of the pharmacokinetic study 





Figure 4 Estimated metabolic pathway of ALC-0159 in various animal species 


in blood (Mo, R) 
in hepatocytes (Mo, R, Mk, H) 
in liver S9 (Mo, R, Mk, H) 





O 
SOY 
Yo ~ i “ N ———— i Me eh et is 
° wWnWwnwanEOr—Oa™°. 
| N,N-ditetradecyl amine 


ALC-0159 mz 440 


H: human, Mk: monkey, Mo: mouse, R: rat 


The major metabolic pathway of ALC-0159 was the hydrolysis of amide bonds to form 
N,N-ditetradecylamine (m/z 410). The metabolites were detected in mouse and rat blood, 


mouse, rat, monkey, and human hepatocytes and liver S9 fractions. 


6. Excretion 

The concentrations of ALC-0315 and ALC-0159 were measured in urine and feces 
collected over time in a PK study in which luciferase RNA-encapsulated LNPs were 
administered intravenously to rats at a dose of 1 mg RNA/kg (Section M2.6.4.3). None of 
the unchanged forms of ALC-0315 or ALC-0159 were detected in the urine. On the other 
hand, unchanged forms of ALC-0315 and ALC-0159 were detected in the feces, and the 
percentages per dose were about 1% and 50%, respectively. As shown in Figure 3, 
metabolites of ALC-0315 were detected in urine. 


7. Pharmacokinetic Drug Interactions 


Pharmacokinetic drug interaction studies have not been conducted for this vaccine. 


8. Other pharmacokinetic studies 
No other pharmacokinetic studies have been conducted for this vaccine. 


9. Discussion and Conclusion 

In the rat PK study, plasma and liver ALC-0315 concentrations decreased to 
approximately 1/7000 and 1/4 of the maximum concentrations, respectively, by 2 weeks 
post-dose, and ALC-0159 concentrations decreased to approximately 1/8000 and 1/250 
of the maximum concentrations, respectively. t% was similar in plasma and liver, 6-8 days 
for LC-0315 and 2-3 days for ALC-0159. The t% was similar in plasma and liver, 6-8 days 
for LC-0315 and 2-3 days for ALC-0159. Plasma t% values may represent the distribution 
of each lipid in the tissues as LNP and its subsequent redistribution in the plasma during 
the elimination process. The unchanged form of ALC-0315 was almost undetectable in 
both urine and feces, but monoester metabolites, double transesterified metabolites, and 
6-hexyl decanoate were detected in feces and plasma samples collected in the rat PK 
study, and glucuronide conjugates of the double transesterified metabolite were 
detected in urine. This metabolic process is thought to be the major mechanism of ALC- 
0315 disappearance, but no quantitative data have been obtained to test this hypothesis. 
On the other hand, about 50% of the dose of ALC-0159 was excreted in feces as 
unchanged drug, and it was metabolized slowly by hydrolysis of amide bond in in vitro 


metabolism experiments. 


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2.6.4 Summary statement of the pharmacokinetic study 





Since the biodistribution of the antigen encoded by BNT162b2 depends on the 
distribution of LNP, we administered luciferase RNA-encapsulated LNP intramuscularly 
to BALB/c mice and examined the biodistribution of alternative reporter proteins. The 
results showed that luciferase was expressed at the site of administration, and was also 
observed in the liver, although at a lower level. The expression of luciferase at the site of 
administration was observed from 6 hours post-dose and disappeared by 9 days post- 
dose. The expression of luciferase in the liver was observed from 6 hours post-dose and 
disappeared by 48 hours post-dose. The distribution in the liver was considered to 
indicate that the locally administered luciferase RNA-encapsulated LNP reached the 
circulating blood and was taken up by the liver. When the radioactivity-conjugated form 
of luciferase RNA-encapsulated LNP was administered intramuscularly to rats, the 
highest radioactivity levels were observed at the site of administration. Outside of the 
dose site, the highest radioactivity was detected in the liver, followed by the spleen, 
adrenal glands, and ovaries, but the total radioactivity recovery relative to the dose in 
these tissues was significantly lower than in the liver. This result is consistent with the 
fact that luciferase expression was observed in the liver in the mouse biodistribution 
study. No toxicity findings indicative of hepatic injury was observed in repeated-dose 


toxicity studies in rats (see Section 2.6.6.3). 


These non-clinical pharmacokinetic evaluations indicated that LNP reaching the 
circulation was distributed in the liver, and the disappearance of ALC-0315 and ALC- 


0159 was related to metabolism and fecal excretion, respectively. 


10. Figures 


Figures are shown in the text and in the summary tables. 


References 

1. World Health Organization. Annex 1. Guidelines on the non-clinical evaluation of 
vaccines. In: WHO Technical Report Series No. 927, Geneva, Switzerland. World 
Health Organization; 2005:31-63. 


2. Guidelines on the non-clinical evaluation of vaccines for the prevention of infectious 
diseases (Pharmaceutical and Food Safety Inspection Service No. 0527-1, May 27, 
2010). 


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2.6.5.1. PHARMACOKINETICS OVERVIEW Test Article: BNT162b2 
Type of Study Test System Test item Method of Testing Facility Report Number 
Administration 
Single Dose Pharmacokinetics 
Single Dose Rat (Wistar Han) modRNA encoding IV bolus Pfizer Inc* PF -07302048 0G 072424 
Pharmacokinetics and luciferase 
Excretion in Urine and Feces formulated in LNP 
of ALC-0159 and ALC-0315 comparable to 
BNT162b2 
Distribution 
In Vivo Distribution Mice BALB/c modRNA encoding IM Injection | R4g-0072 
luciferase 
formulated in LNP 
comparable to 
BNT162b2 
In Vivo Distribution Rat (Wistar Han) modRNA encoding IM Injection a: 185350 
luciferase 
formulated in LNP 
comparable to 
BNT162b2 with 
trace amounts of 
[>H]-CHE as non- 
diffusible label 
Metabolism 
In Vitro and In Vivo Metabolism 
In Vitro Metabolic Stability Mouse (CD-1/ICR), rat ALC-0315 In vitro 01049008 
of ALC-0315 in Liver (Sprague Dawley and 
Microsomes Wistar Han), monkey 
(Cynomolgus), and 
human liver microsomes 
In Vitro Metabolic Stability Mouse (CD-1/ICR), rat ALC-0315 In vitro 01049009 
of ALC-0315 in Liver S9 (Sprague Dawley), 
monkey (Cynomolgus), 
and human S9 liver 
fractions 
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2.6.5 FSD) ReR ER D EES 



















































































2.6.5.1. 


Type of Study 


Test System 


PHARMACOKINETICS OVERVIEW 


Testing Facility 


Test Article: BNT162b2 


Report Number 








In Vitro Metabolic Stability 
of ALC-0315 in Hepatocytes 


In Vitro Metabolic Stability 


of ALC-0159 in Liver 
Microsomes 


In Vitro Metabolic Stability 


of ALC-0159 in Liver S9 


In Vitro Metabolic Stability 
of ALC-0159 in Hepatocytes 


Biotransformation of 


ALC-0159 and ALC-0315 In 


Vitro and In Vivo in Rats 


Mouse (CD-1/ICR), rat 
(Sprague Dawley and 
Wistar Han), monkey 

(Cynomolgus), and 
human hepatocytes 

Mouse (CD-1/ICR), rat 
(Sprague Dawley and 
Wistar Han), monkey 

(Cynomolgus), and 
human liver microsomes 

Mouse (CD-1/ICR), rat 

(Sprague Dawley), 
monkey (Cynomolgus), 
and human S9 fractions 

Mouse (CD-1/ICR), rat 
(Sprague Dawley and 
Wistar Han), monkey 

(Cynomolgus), and 
human hepatocytes 
In vitro: 

CD-1 mouse, Wistar 
Han rat, cynomolgus 
monkey, and human 
blood, liver S9 fractions 
and hepatocytes 
In vivo: male Wistar Han 
rats 





Pfizer Inc® 


01049010 


01049020 


01049021 


01049022 


PF-07302048 OSM 043725 





Test item Method of 
Administration 
ALC-0315 In vitro 
ALC-0159 In vitro 
ALC-0159 In vitro 
ALC-0159 In vitro 
ALC-0315 and In vitro or 
ALC-0159 IV (in vivo in 
rats) 
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2.6.5.1. PHARMACOKINETICS OVERVIEW Test Article: BNT162b2 


Type of Study Test System Test item Method of Testing Facility Report Number 
Administration 





ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an excipient in the LNP formulation 
used in BNT162b2; ALC-0315 = (4-hydroxybutyl)azanediyl)bis(hexane-6, | -diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the 
LNP formulation used in BNT162b2; IM = Intramuscular; IV = Intravenous; LNP = lipid nanoparticles; S9 = Supernatant fraction obtained from liver 
homogenate by centrifuging at 9000 g. 

a. La Jolla, California. 


b. , Germany. 
Cc. , UK. 
d. , China. 


e. Groton, Connecticut. 


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2.6.5.3. PHARMACOKINETICS: Test Article: modRNA encoding luciferase in LNP 
PHARMACOKINETICS AFTER A SINGLE DOSE Report Number: PF-07302048_0g{l 072424 
Species (Strain) Rat (Wistar Han) 

Sex/Number of Animals Male/ 3 animals per timepoint® 

Feeding Condition Fasted 

Method of Administration IV 

Dose modRNA (mg/kg) 1 

Dose ALC-0159 (mg/kg) 1.96 

Dose ALC-0315 (mg/kg) 15.3 

Sample Matrix Plasma, liver, urine and feces 

Sampling Time Points (h post dose): Predose, 0.1, 0.25, 0.5, 1, 3, 6, 24, 48, 96, 192, 336 

Analyte ALC-0315 ALC-0159 

PK Parameters: Mean? Mean? 

AUCine (ugeh/mL)° 1030 99.2 

AUCrast (ugeh/mL) 1020 98.6 

Initial ty, (h)4 1.62 1.74 

Terminal elimination ty, (h)° 139 d2eih 

Estimated fraction of dose distributed to liver (%)! 59.5 20.3 

Dose in Urine (%) NC& NC® 

Dose in Feces (%)" 1.05 47.2 





ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an excipient in the LNP formulation 
used in BNT162b2; ALC-0315 = (4-hydroxybutyl)azanediyl)bis(hexane-6, | -diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the 
LNP formulation used in BNT162b2; AUCinr = Area under the plasma drug concentration-time curve from 0 to infinite time; AUCtast = Area under the plasma 
drug concentration-time curve from 0 to the last quantifiable time point; BLQ = Below the limit of quantitation; LNP = Lipid nanoparticle; 

modRNA = Nucleoside modified messenger RNA; PK = Pharmacokinetics; ty,= Half-life. 

a. Non-serial sampling, 36 animals total. 

b. Only mean PK parameters are reported due to non-serial sampling. 

c. Calculated using the terminal log-linear phase (determined using 48, 96, 192, and 336 h for regression calculation). 

d. In(2)/initial elimination rate constant (determined using 1, 3, and 6 h for regression calculation). 

e. In(2)/terminal elimination rate constant (determined using 48, 96, 192, and 336 h for regression calculation). 

f. Calculated as follows: highest mean amount in the liver (ug)/total mean dose (ug) of ALC-0315 or ALC-0159. 

g. Not calculated due to BLQ data. 

h. Fecal excretion, calculated as: (mean ug of analyte in feces/ mean pg of analyte administered) x 100 


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2.6.5.55A. PHARMACOKINETICS: ORGAN 
DISTRIBUTION 


Test Article: modRNA encoding luciferase in LNP 
Report Number: R§-0072 








Species (Strain): Mice (BALB/c) 
Sex/Number of Animals: Female/3 per group 
Feeding Condition: Fed ad libitum 
Vehicle/Formulation: Phosphate-buffered saline 
Method of Administration: Intramuscular injection 
Dose (mg/kg): 1 pg/hind leg in gastrocnemius muscle (2 pg total) 
Number of Doses: 1 
Detection: Bioluminescence measurement 
Sampling Time (hour): 6, 24, 48, 72 hours; 6 and 9 days post-injection 
Time point Total Mean Bioluminescence signal (photons/second) Mean Bioluminescence signal in 
the liver (photons/second) 
Buffer control modRNALuciferase in LNP modRNALuciferase in LNP 
6 hours 1.28x10° 1.26x10° 4.94x107 
24 hours 2.28x10° 7.31x108 2.4x10° 
48 hours 1.40x10° 2.10108 Below detection* 
72 hours 1.33x10° 7.87x107 Below detection* 
6 days 1.62x10° 2.92x10° Below detection® 
9 days 7.66104 5.09x10° Below detection® 





LNP = Lipid nanoparticle; modRNA = Nucleoside modified messenger RNA. 


a. At or below the background level of the buffer control. 


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2.6.5.5B. PHARMACOKINETICS: ORGAN Test Article: [*>H]-Labelled LNP-mRNA formulation containing 
DISTRIBUTION CONTINUED ALC-0315 and ALC-0159 
Report Number: 185350 
Species (Strain): Rat (Wistar Han) 
Sex/Number of Animals: Male and female/3 animals/sex/timepoint (21 animals/sex total for the 50 ug dose) 
Feeding Condition: Fed ad libitum 
Method of Administration: Intramuscular injection 
Dose: 50 wg [H]-08-A01-CO (lot # NC-0552-1) 
Number of Doses: 1 
Detection: Radioactivity quantitation using liquid scintillation counting 
Sampling Time (hour): 0.25, 1, 2, 4, 8, 24, and 48 hours post-injection 
Sample Mean total lipid concentration (ug lipid equivalent/g (or mL) % of administered dose (males and females combined) 
(males and females combined) 
0.25h lh 2h 4h 8h 24h 48h 0.25 h 1h 2h 4h 8h 24h 48h 
Adipose tissue 0.057 0.100 0.126 0.128 0.093 0.084 0.181 -- -- -- -- -- -- -- 
Adrenal glands 0.271 1.48 2.72 2.89 6.80 13.8 18.2 0.001 0.007 0.010 0.015 0.035 0.066 0.106 
Bladder 0.041 0.130 0.146 0.167 0.148 0.247 0.365 0.000 0.001 0.001 0.001 0.001 0.002 0.002 
Bone (femur) 0.091 0.195 0.266 0.276 0.340 0.342 0.687 -- -- -- -- -- = -- 
Bone marrow 0.479 0.960 1.24 1.24 1.84 2.49 3.77 -- -- -- -- -- -- -- 
(femur) 
Brain 0.045 0.100 0.138 0.115 0.073 0.069 0.068 0.007 0.013 0.020 0.016 0.011 0.010 0.009 
Eyes 0.010 0.035 0.052 0.067 0.059 0.091 0.112 | 0.000 0.001 0.001 0.002 0.002 0.002 0.003 
Heart 0.282 1.03 1.40 0.987 0.790 0.451 0.546 | 0.018 0.056 0.084 0.060 0.042 0.027 0.030 
Injection site 128 394 311 338 213 195 165 19.9 52.6 31.6 28.4 21.9 29.1 24.6 
Kidneys 0.391 1.16 2.05 0.924 0.590 0.426 0.425 0.050 0.124 0.211 0.109 0.075 0.054 0.057 
Large intestine 0.013 0.048 0.093 0.287 ~=0.649 1.10 1.34 0.008 0.025 0.065 0.192 0.405 0.692 0.762 
Liver 0.737 4.63 11.0 16.5 26.5 19.2 24.3 0.602 2.87 7.33 11.9 18.1 15.4 16.2 
Lung 0.492 1.21 1.83 1.50 1.15 1.04 1.09 0.052 0.101 0.178 0.169 0.122 0.101 0.101 








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2.6.5.5B. PHARMACOKINETICS: ORGAN 
DISTRIBUTION CONTINUED 


Test Article: [7>H]-Labelled LNP-mRNA formulation containing 
ALC-0315 and ALC-0159 
Report Number: 185350 














Sample Total Lipid concentration (ug lipid equivalent/g [or mL]) % of Administered Dose (males and females combined) 
(males and females combined) 
0.25h lh 2h 4h 8h 24h 48h 0.25 h lh 2h 4h 8h 24h 48h 
Lymph node 0.064 0.189 0.290 0.408 0.534 0.554 0.727 -- -- -- -- -- -- -- 
(mandibular) 
Lymph node 0.050 0.146 0.530 0.489 0.689 0.985 1.37 -- -- -- -- -- -- -- 
(mesenteric) 
Muscle 0.021 0.061 0.084 0.103 0.096 0.095 0.192 -- -- = -- -- -- -- 
Ovaries 0.104 1.34 1.64 2.34 3.09 5.24 12.3 0.001 0.009 0.008 0.016 0.025 0.037 0.095 
(females) 
Pancreas 0.081 0.207 ~=—0.414 0.380 0.294 0.358 0.599 | 0.003 0.007 0.014 0.015 0.015 0.011 0.019 
Pituitary gland 0.339 0.645 0.868 0.854 0.405 0.478 0.694 | 0.000 0.001 0.001 0.001 0.000 0.000 0.001 
Prostate 0.061 0.091 0.128 0.157. 0.150 0.183 0.170 | 0.001 0.001 0.002 0.003 0.003 0.004 0.003 
males 
ae 0.084 0.193 0.255 0.220 0.135 0.170 0.264 | 0.003 0.007 0.008 0.008 0.005 0.006 0.009 
glands 
Skin 0.013 0.208 0.159 0.145 0.119 0.157 0.253 -- -- -- -- -- -- -- 
Small intestine 0.030 0.221 0.476 0.879 1.28 1.30 1.47 0.024 0.130 0.319 0.543 0.776 0.906 0.835 
Spinal cord 0.043 0.097 ~—0.169 0.250 0.106 0.085 0.112 | 0.001 0.002 0.002 0.003 0.001 0.001 0.001 
Spleen 0.334 2.47 7.73 10.3 22.1 20.1 23.4 0.013 0.093 0.325 0.385 0.982 0.821 1.03 
Stomach 0.017 0.065 0.115 0.144 0.268 0.152 0.215 | 0.006 0.019 0.034 0.030 0.040 0.037 0.039 
Testes (males) 0.031 0.042 0.079 0.129 0.146 0.304 0.320 | 0.007 0.010 0.017 0.030 0.034 0.074 0.074 
Thymus 0.088 0.243 =—-0.340 0.335 0.196 0.207 0.331 0.004 0.007 0.010 0.012 0.008 0.007 0.008 
Thyroid 0.155 0.536 0.842 0.851 0.544 0.578 = 1.00 0.000 0.001 0.001 0.001 0.001 0.001 0.001 
Uterus 0.043 0.203 0.305 0.140 0.287 0.289 0.456 | 0.002 0.011 0.015 0.008 0.016 0.018 0.022 
(females) 
Whole blood 1.97 4.37 5.40 3.05 1.31 0.909 0.420 -- -- -- -- -- -- -- 
Plasma 3.97 8.13 8.90 6.50 2.36 1.78 0.805 -- = -- -- -- -- -- 
Blood:Plasma 0.815 0.515 0.550 0.510 0.555 0.530 0.540 -- -- -- -- -- -- -- 
ratio* 
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2.6.5.5B. PHARMACOKINETICS: ORGAN Test Article: [7H]-Labelled LNP-mRNA formulation containing 


DISTRIBUTION CONTINUED ALC-0315 and ALC-0159 
Report Number: 185350 





-- = Not applicable, partial tissue taken; [>H]-08-A01-CO = An aqueous dispersion of LNPs, including ALC-0315, ALC-0159, distearoylphosphatidylcholine, 
cholesterol, mRNA encoding luciferase and trace amounts of radiolabeled [Cholesteryl-1,2-3H(N)]-Cholesteryl Hexadecyl Ether, a nonexchangeable, non- 
metabolizable lipid marker used to monitor the disposition of the LNPs; ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N--ditetradecylacetamide), a proprietary 
polyethylene glycol-lipid included as an excipient in the LNP formulation used in BNT162b2; ALC-0315 = (4--hydroxybutyl)azanediyl)bis(hexane-6, | - 
diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the LNP formulation used in BNT162b2; LNP = Lipid nanoparticle; 

mRNA = messenger RNA. 

a. The mean male and female blood:plasma values were first calculated separately and this value represents the mean of the two values. 


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2.6.5.9. PHARMACOKINETICS: METABOLISM IN VIVO, 


RAT 


Species (Strain): 

Sex/ Number of animals 

Method of Administration: 

Dose (mg/kg): 

Test System: 

Analysis Method: 
Biotransformation 


N-dealkylation, oxidation 
N-Dealkylation, oxidation 
N-dealkylation, oxidation 
N-Dealkylation, oxidation 
N-dealkylation, hydrolysis, oxidation 
Hydrolysis (acid) 

Hydrolysis, hydroxylation 
Bis-hydrolysis (amine) 

Hydrolysis, glucuronidation 
Bis-hydrolysis (amine), glucuronidation 
Bis-hydrolysis (amine), glucuronidation 
Hydrolysis (amine) 

Hydrolysis (amine), Glucuronidation 
Oxidation to acid 

Oxidation to acid 

Hydroxylation 

Sulfation 

Sulfation 

Glucuronidation 

Glucuronidation 


m/z 


102.0561? 
104.0706° 
130.0874? 
132.1019° 
145.0506 
255.2330" 
271.2279" 
290.2690° 
431.2650 
464.2865 
466.3011 
528.4986 
704.5307 
778.6930 
780.7076 
782.7232 
844.6706 
846.6851 
940.7458 
942.7604 





Tap TF ap TFT TFT PF TF TF BP PB 


Test Article: modRNA encoding luciferase in LNP 








Report Number: PF-07302048_0 | 043725 
Rat (Wistar Han) 
Male/ 36 animals total for plasma and liver, 3 animals for urine and feces 
Intravenous 
1 
Plasma, Urine, Feces, Liver 
Ultrahigh performance liquid chromatography/ mass spectrometry 
Metabolites of ALC-0315 Detected 
Plasma | Urine Feces | Liver 

ND ND ND ND 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 

+ ND ND ND 
ND ND ND ND 

+ + + + 
ND ND ND ND 
ND ND ND ND 
ND + ND ND 

cb ND ND =H: 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 
ND ND ND ND 





























Note: Both theoretical and observed metabolites are included. 


m/z = mass to charge ratio; ND = Not detected; + = minor metabolite as assessed by ultraviolet detection. 


a. Negative ion mode. 
b. Positive ion mode. 


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Test Article: ALC-0315 
Report Numbers: 01049 


2.6.5.10A. PHARMACOKINETICS: METABOLISM IN VITRO 




















Type of Study: Stability of ALC-0315 In Vitro 
Study System: Liver Microsomes + NADPH S9 Fraction + NADPH, UDPGA, and Hepatocytes 
alamethicin 
ALC-0315 1 uM 1 uM 1 uM 
Concentration: 
Duration of 120 min 120 min 240 min 
Incubation (min): 
Analysis Method: Ultra-high performance liquid chromatography-tandem mass spectrometry 
Incubation time Percent ALC-0315 remaining 
(min) Liver Microsomes Liver S9 Fraction Hepatocytes 
Mouse Rat Rat Monkey Human Mouse Rat(SD) Monkey Human | Mouse Rat Rat Monkey Human 
(CD- (SD) (WH) (Cyno) (CD- (Cyno) (CD- (SD) (WH) = (Cyno) 
1/ICR) 1/ICR) 1/ICR) 
0 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 
15 98.77 94.39 96.34 97.96 100.24 97.69 98.85 99.57 95.99 -- -- -- -- -- 
30 97.78 96.26 97.32 96.18 99.76 97.22 99.62 96.96 97.32 101.15 97.75 102.70 96.36 100.72 
60 100.49 = 99.73 98.54 100.00 101.45 98.61 99.62 99.13 94.98 100.77 98.50 102.32 97.82 101.44 
90 97.78 98.66 94.15 97.96 100.48 98.15 98.85 98.70 98.33 101.92 99.25 103.09 100.0 100.36 
120 96.54 95.99 93.66 97.71 98.31 96.76 98.46 99.57 99.33 98.85 97.38 99.61 96.36 100.72 
180 -- -- -- -- -- -- -- -- -- 101.15 98.88 103.47 95.64 98.92 
240 -- - -- -- -- -- -- -- -- 99.62 101.12 100.00 93.82 99.64 
ty (min) >120 >120 >120 >120 >120 >120 >120 >120 >120 >240 >240 >240 >240 >240 














-- = Data not available; ALC-0315 = (4-hydroxybutyl)azanediyl)bis(hexane-6, | -diyl)bis(2-hexyldecanoate), a proprietary aminolipid included as an excipient in the lipid 


nanoparticle formulation used in BNT162b2; Cyno = Cynomolgus; NADPH = Reduced form of nicotinamide adenine dinucleotide phosphate; NC = not calculated; SD = Sprague 
Dawley; ty = half-life; WH = Wistar-Han; UDPGA= uridine-diphosphate-glucuronic acid trisodium salt. 


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Test Article: ALC-0159 
Report Numbers: 01049 


2.6.5.10B. PHARMACOKINETICS: METABOLISM IN VITRO 
CONTINUED 





Type of Study: Stability of ALC-0159 In Vitro 

Study System: Liver Microsomes + NADPH S9 Fraction + NADPH, UDPGA, and Hepatocytes 
alamethicin 

ALC-0159 1 uM 1 uM 1 uM 

Concentration: 

Duration of 120 min 120 min 240 min 


Incubation (min): 











Analysis Method: Ultra-high performance liquid chromatography-tandem mass spectrometry 
Incubation time Percent ALC-0159 remaining 
(min) Liver Microsomes Liver S9 Fraction Hepatocytes 
Mouse Rat Rat Monkey Human Mouse Rat (SD) Monkey Human | Mouse Rat Rat Monkey Human 
(CD- (SD) (WH) (Cyno) (CD-1/ICR) (Cyno) (CD- (SD) (WH) (Cyno) 
1/ICR) 1/ICR) 
0 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 
15 82.27 101.24 =112.11 100.83 99.59 98.93 84.38 91.30 106.73 -- -- -- -- -- 
30 86.40 93.78 102.69 85.12 92.28 91.10 90.87 97.96 107.60 100.85 93.37 113.04 90.23 106.34 
60 85.54 98.34 105.38 86.36 95.53 102.85 97.97 105.56 104.97 94.92 91.81 105.07 92.93 101.58 
90 85.41 95.44 100.90 94.63 97.97 90.75 93.51 108.33 109.36 94.28 90.25 112.80 94.59 92.67 
120 95.87 97.10 108.97 93.39 93.09 106.76 92.70 105.74 119.59 87.08 89.47 104.11 97.51 96.04 
180 -- -- -- -- -- -- -- -- -- 94.92 93.96 102.90 89.81 93.66 
240 -- -- -- -- -- -- -- -- -- 102.75 94.93 98.79 92.93 102.57 
ty, (min) >120 >120 >120 >120 >120 >120 >120 >120 >120 >240 >240 >240 >240 >240 














-- = Data not available; ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), a proprietary polyethylene glycol-lipid included as an excipient in the lipid 
nanoparticle formulation used in BNT162b2; Cyno = Cynomolgus; NADPH = Reduced form of nicotinamide adenine dinucleotide phosphate; NC = not calculated; SD = Sprague 
Dawley; WH = Wistar-Han; UDPGA= uridine-diphosphate-glucuronic acid trisodium salt. 


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2.6.5.10C. PHARMACOKINETICS: METABOLISM Test Article: ALC-0315 
IN VITRO CONTINUED Report Number: PF-07302048 0S 043725 
Type of study Metabolism of ALC-0315 In Vitro 
Study system Blood Hepatocytes Liver S9 Fraction 
ALC-0315 concentration 10 uM 10 uM 10 uM 
Duration of incubation 24h 4h 24h 
Analysis Method: Ultrahigh performance liquid chromatography/ mass spectromet 
Biotransformation Hepatocytes Liver S9 Fraction 

Mouse | Rat | Monkey | Human | Mouse Rat Monkey | Human Mouse | Rat | Monkey Human 
N-dealkylation, oxidation 102.0561# ND ND ND ND ND ND ND ND ND ND ND ND 
N-Dealkylation, oxidation 104.0706" ND ND ND ND ND ND ND ND ND ND ND ND 
N-dealkylation, oxidation 130.0874 ND ND ND ND ND ND ND ND ND ND ND ND 
N-Dealkylation, oxidation 132.1019° ND ND ND ND ND ND ND ND ND ND ND ND 
N-dealkylation, hydrolysis, oxidation 145.0506 ND ND ND ND ND ND ND ND ND ND ND ND 
Hydrolysis (acid) 255.2330? ND ND ct + + | ND + 
Hydrolysis, hydroxylation 271.2279 ND ND ND ND ND ND ND ND ND ND ND ND 
Bis-hydrolysis (amine) 290.2690 ND ND ND ND ND ND ND ND + ND 
Hydrolysis, glucuronidation 431.2650? ND ND ND ND ND ND ND ND ND ND ND ND 
Bis-hydrolysis (amine), glucuronidation 464.2865? ND ND ND ND ND ND ND ND ND ND ND ND 
Bis-hydrolysis (amine), glucuronidation 466.3011 ND ND ND ND ND ND ND ND ND ND ND ND 
Hydrolysis (amine) 528.4986 ND + ND ND ND ND ND ND ND ND + ND 
Hydrolysis (amine), glucuronidation 704.5307 ND ND ND ND ND ND ND ND ND ND ND ND 
Oxidation to acid 778.6930 ND ND ND ND ND ND ND ND ND ND ND ND 
Oxidation to acid 780.7076 ND ND ND ND ND ND ND ND ND ND ND ND 
Hydroxylation 782.7232 ND ND ND ND ND ND ND ND ND ND ND ND 
Sulfation 844.67067 ND ND ND ND ND ND ND ND ND ND ND ND 
Sulfation 846.6851 ND ND ND ND ND ND ND ND ND ND ND ND 
Glucuronidation 940.7458 ND ND ND ND ND ND ND ND ND ND ND ND 
Glucuronidation 942.7604 ND ND ND ND ND ND ND ND ND ND ND ND 
Note: Both theoretical and observed metabolites are included. 
m/z = mass to charge ratio; ND = Not detected; + = metabolite present. 
a. Negative ion mode. 
b. Positive ion mode. 

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2.6.5.10D. PHARMACOKINETICS: METABOLISM Test Article: ALC-0159 
IN VITRO CONTINUED Report Number: PF-07302048 05MM 043725 
Type of study Metabolism of ALC-0159 In Vitro 
Study system Blood Hepatocytes Liver S9 Fraction 
ALC-0159 concentration 10 uM 10 uM 10 uM 
Duration of incubation 24h 4h 24h 
Analysis Method: Ultrahigh performance liquid chromatography/ mass spectromet 
Biotransformation Hepatocytes Liver S9 Fraction 
Mouse | Rat | Monkey | Human | Mouse Rat Monkey | Human | Mouse | Rat | Monkey | Human 
O-Demethylation, O-dealkylation 107.0703 ND ND ND ND ND ND ND ND ND ND ND ND 
O-Demethylation, O-dealkylation 151.0965 ND ND ND ND ND ND ND ND ND ND ND ND 
O-Demethylation, O-dealkylation 195.1227 ND ND ND ND ND ND ND ND ND ND ND ND 
Hydrolysis, N-Dealkylation 214.2529 ND ND ND ND ND ND ND ND ND ND ND ND 
N-Dealkylation, oxidation 227.20178 ND ND ND ND ND ND ND ND ND ND ND ND 
Hydrolysis (amine) 410.4720 + + ND ND + + + 
N,N-Didealkylation 531.5849 ND ND ND ND ND ND ND ND ND ND ND ND 
N-Dealkylation 580.6396 ND ND ND ND ND ND ND ND ND ND ND ND 
O-Demethylation, oxidation 629.6853 ND ND ND ND ND ND ND ND ND ND ND ND 
Hydroxylation 633.6931 ND ND ND ND ND ND ND ND ND ND ND ND 
@-Hydroxylation, Oxidation 637.1880 ND ND ND ND ND ND ND ND ND ND ND ND 
Hydrolysis (acid) 708.7721 ND ND ND ND ND ND ND ND ND ND ND ND 
Note: Both theoretical and observed metabolites are included. 
m/z = mass to charge ratio; ND = Not detected; + = metabolite present. 
a. Negative ion mode. 
b. Positive ion mode. 
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